Although there exists extensive information indicating that membrane alterations are involved in the neoplastic process, very little is known concerning the role of phospholipid and sterol alterations in neoplasia, especially in the formation of metastasis. The aim of this research is to investigate in depth the structure and functions of metastatic cell membranes. The role of membrane bilayer lipid asymmetry utilizing tumorigenic cell lines in culture (LM cell variants and B-16 melanoma variants) will be extended to study of the same cell lines growing as primary tumors and as metastatic tumors in mice. We recently have established an in vivo model for a line of mouse fibroblasts, LM cells, which grow in vitro in chemically defined serum-free media. Inoculation of LM fibroblasts subcutaneously into athymic (nude) mice produces malignant fibrosarcomas which metastasize to lungs. Preliminary data indicate that membrane characteristics of the metastases such as Na+,K+-ATPase, benzodiazepine receptor binding, phase transitions, and fluidity gradients, were markedly different from those of primary tumor membranes. These differences were maintained in culture. The aminophospholipid composition and asymmetric distribution in plasma membranes of LM fibroblasts injected s.c. was correlated with the appearance of spontaneous lung metastases in the nude mice. The transbilayer distribution of phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and sterols in each tumor cell system will be determined by utilizing chemical labeling reagents, phospholipid exchange proteins, and fluorescent sterol analogs. Phase alterations and fluidity gradients will be studied by use of fluorescence probes. Transmembrane functions will be evaluated by measuring cell membrane-membrane contact (agglutination), transport (thymidine, deoxyglucose, and Na+,K+-ATPase) and macromolecular uptake phagocytosis of latex beads and pinocytosis of horseradish peroxidase). Basic biochemical and biological mechanisms involved with methylation of phospholipid (benzodiazepine receptor) and of transmembrane processes (Na+,K+-ATPase and prostaglandin-mediated adenylate cyclase) will be investigated in detail and correlated with alterations in membrane lipid asymmetry in the metastatic cell membranes as compared to primary tumor cell plasma membranes both in vivo and in culture. The biochemical, chemical, biophysical and pathological results will be correlated to determine if alterations in membrane lipid asymmetry of several tumorigenic cell lines are associated with enhanced tumorigenicity and metastatic capability in the animal models.